The green fluorescent protein (GFP) from the jellyfish Aequorea Victoria has provided a myriad of applications for biological systems (Tsien, R. Y. (1998) Ann. Rev. Biochem. 67, 509-544). Over the last several years, both random and semi-rational mutagenesis have produced GFP variants with new colors, improved folding property, more brightness, and altered pH-sensitivity. Through genetic manipulations, hundreds of proteins have been successfully fused to GFPs to allow monitoring of their expression and trafficking. When GFP or GFP-fusion protein is heterologously expressed at a certain level, the intensity of the fluorescence depends on:    (1) the ultimate brightness of GFP fluorophore, which is limited by the product of extinction coefficient (ε) and fluorescence quantum yield (Φ).    (2) the maturation efficiency of newly-synthesized GFP polypeptides.    (3) the extent of quenching of GFP fluorophore by environmental factors.
The yellow fluorescent protein (YFP) is one of the most commonly used GFP variants and has the longest-wavelength emission of all Aequorea GFP variants. The ε and Φ of most YFP variants are within 60,000 to 100,000 M−1 cm−1 and 0.6 to 0.8, respectively (Tsien, R. Y. (1998) Ann. Rev. Biochem. 67, 509-544). These values are almost comparable to those of common bright fluorophores, such as fluorescein and rhodamine. Therefore, the improvement of the ultimate brightness of YFP seems to have reached its limit.
Newly-synthesized GFP polypeptides need to mature properly before emitting fluorescence. The maturation involves two steps: first, the protein folding into a nearly native conformation, and then, cyclization of an internal tripeptide followed by oxidation. Some of the primary mutations that improve maturation of GFP have been identified (Tsien, R. Y. (1998) Ann. Rev. Biochem. 67, 509-544). For example, F64L/M153T/V163A/S175G are common mutations introduced in many enhanced GFP variants. M153T and S175G are located on the surface of the β-barrel and are known to enhance the folding efficiency and the stability by reducing surface hydrophobicity and increasing the solubility of the protein. However, no mutation has been clearly identified to facilitate the final oxidation reaction at 37° C., even though such mutations are very desirable since the oxidation step is the rate-limiting step in the whole process of GFP maturation. Although there has been some improvement in the folding efficiency, slow maturation is still one of the biggest problems in the application of GFP variants for visualization and is even more problematic when they are expressed at 37° C. and/or targeted to certain organelles. Therefore, obtaining GFP variants that have better maturation efficiency is very crucial and necessary at this stage.
Among Aequorea GFP variants, YFPs are relatively acid sensitive, and uniquely quenched by halide ions, including chloride (Cl−) (Jayaraman, S., et al. (2000) J. Biol. Chem. 275, 6047-6050; Wachter, R. M., et al. (2000) J. Mol. Biol. 301, 157-171). Proton (H+) and C− synergistically affect the charge state of the chromophore of YFP, thereby suppressing the fluorescence. The concentrations of these ions vary among intracellular organelles; there is a significant accumulation of H+ in secretory organelles. In addition, their concentrations change dynamically upon stimulations (Kuner, T. et al (2000) Neuron 27, 447-459). For instance, glutamate and electrical stimulations of hippocampal neurons reduce intracellular pH by about 0.4, and the receptor-mediated Cl− fluxes occur in olfactory and GABAergic neurons. To let YFP be fully and stably fluorescent in these situations, the mutations that decrease the sensitivity to both H+ and Cl− are desired.